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PubMed2004

Progress in plant metabolic engineering.

Over the past few years, there has been a growing realization that metabolic pathways must be studied in the context of the whole cell rather than at the single pathway level, and that even the simplest modifications can send ripples throughout the entire system. Attention has therefore shifted away from reductionist, single-gene engineering strategies and towards more complex approaches involving the simultaneous overexpression and/or suppression of multiple genes. The use of regulatory factors to control the abundance or activity of several enzymes is also becoming more widespread. In combination with emerging methods to model metabolic pathways, this should facilitate the enhanced production of natural products and the synthesis of novel materials in a predictable and useful manner.

Alkaloids↗
PubMed2004

[Chimeric T cell receptor N29gamma redirect T lymphocytes response specific to p185HER2 in A murine model of metastatic breast cancer].

BACKGROUND & OBJECTIVE: Chimeric T-cell receptors (chTCR) are recombinant immune receptors with the characteristics of combining exquisite antigen specificity of a monoclonal antibody and activating T lymphocyte function by signal transduction element. Compared to "classic" TCR in mediating T cells immune response to target cells,a significant potential advantage of chTCR is the lack of major histocompatibility complex (MHC) restriction and antigen processing. N29gamma is a chTCR specific for p185HER2. The current study is to investigate the efficacy of N29gamma redirect T cells in response to p185HER2 in a murine model of metastatic breast cancer. METHODS: Splenic T cells from Balb/c mice were purificated by negative selection over sterile brushed nylon wool fiber,activated by immobilized purified anti-mouse CD3 and CD28 antibodies. Then the recombinant retrovirus pRet6N29gamma were transduced by centrifuging (1 300 g for 90 min, at 32 Centigrade). The transduction efficiency was indirectly defermined by green fluorescence protein (GFP) expression of T cells in control group tested by flow cytometry. Mice bearing 3 days or 8 days MT901 or MT901/HER2 were randomized into experiment group or control group to receive IV infusions of polyclonal activated Balb/c splenic T cells transduced with the N29gamma retroviral vector or a control GFP vector(5 x 10(6)-40 x 10(6) of T cells per mouse). Each mouse was injected intraperitoneal with IL-2 of 3 x 10(4) IU every 12 h for 10 times. Mice were sacrificed at 11 days (Day 3 model) or 13 days (Day 8 model) after T cells transfer to enumerate the lung metastases. RESULTS: In Day 3 model, more than 200 lung metastases were present in mice in mock group, received non-transduced T cells group as well as GFP transduced T cells group. Treatment of mice bearing HER2 positive tumor with N29gamma transduced T cells resulted in a significant reduction in the number of lung metastases (3.4+/-3.3/lung). In contrast,N29gamma transduced T cells failed to induce the regression of HER2 negative MT901 pulmonary metastases (>200/lung). In Day 8 model, treatment with N29gamma modified T cells had a cell dose dependent effect on HER2+ lung metastases. Compared with 4 x 10(7) GFP T cells, 107 of N29gamma T cells were not enough to reduce the number of lung metastases (167+/-15.3) (P=0.198). With increasing dosage of N29gamma modified T cells from 2,3 to 4 x 10(7),there was a progressive decrease in the number of pulmonary metastases from 64+/-12.1,34+/-6.3,to 8+/-4.3,respectively (P< 0.01). CONCLUSIONS: Gene engineered T cells expressing chimeric TCR N29gamma induced the HER2-specific regression of lung metastases in breast cancer. Higher doses of gene-modified T cells were necessary to eliminate more advanced lung metastases.

Animals↗
PubMed1996

A modular set of Flp, FRT and lacZ fusion vectors for manipulating genes by site-specific recombination.

Site-specific recombinases can serve as powerful tools to target genetic manipulations to specific cell populations in culture and in the organism. A series of vectors for engineering gene activation, deletion and integration in mammalian cells using Flp recombinase is described here. The vectors are modular in design so that specific cassettes can be linked depending on the application. Using these vectors, efficient Flp-mediated lacZ activation and beta-galactosidase (beta Gal) detection has been demonstrated in mammalian cell culture. These vectors should facilitate using Flp to mark cell populations, as well as to activate, remove or mutate genes in culture and in the mouse.

Amino Acid Sequence↗
PubMed2005

Cell death induced by the herpes simplex virus-1 thymidine kinase delivered by human immunodeficiency virus-1-based virus-like particles.

HIV-1 Nef incorporates into virions at low levels, likely about 10 molecules per viral particle. Here, we describe a Nef mutant (Nef7) apparently showing more than 100-fold higher efficiency of virion incorporation. Interestingly, Nef7 can act as a cargo molecule for protein delivery into the cells, as its virion incorporation appeared conserved even upon C-terminal fusion with proteins of up to 30 kDa. This was demonstrated first by assessing the intracellular fluorescence of cells challenged with lentivirus-based virus-like particles (VLPs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) and incorporating Nef7 fused with the green fluorescent protein. Furthermore, the biologic activity of products delivered by Nef7-based VLPs was demonstrated by tagging Nef7 with the herpes simplex virus-1 thymidine kinase (HSV-1 TK). In fact, we observed that both cell lines and primary human macrophages challenged with (VSV-G) Nef7/TK VLPs died after 5 to 7 days of treatment with ganciclovir (GCV). In sum, our findings support the notion that Nef7-based VLPs can be considered platforms for original systems of protein delivery. In particular, the here- described Nef7/TK VLPs represent a first applicative example opening the way toward new HSV-1 TK/GCV-based cell suicide therapies circumventing cell gene engineering procedures.

Blotting, Western↗
PubMed2006

A highly efficient polyethylene glycol-mediated transformation method for mushrooms.

A highly efficient transformation system mediated by polyethylene glycol was developed for the cultivated mushroom Pleurotus ostreatus. Eighty to 180 integrative and stable-resistant colonies appeared per mug of DNA per 10(7) viable protoplasts in a transformation experiment with the hygromycin B phosphotransferase gene (hph), which is about 40-1800 times higher than that previously reported in P. ostreatus. One hundred to 150 transformants emitting green fluorescence were observed per mug of DNA per 10(7) viable protoplasts in a transformation with the green fluorescent protein gene, but green fluorescence disappeared 30 h after transformation, suggesting that the green fluorescent protein gene was only transiently expressed in P. ostreatus. Plasmid pAN7-1 was also transferred into two important cultivated mushrooms, Ganoderma lucidum and Lentinus edodes, and 120-150 and 85-100 transformants per mug of DNA per 10(7) viable protoplasts were obtained, respectively, which is seven to 38 times and 24-28 times greater than previously reported. These data indicate that this new polyethylene glycol-mediated transformation procedure is highly efficient for mushrooms, and could be a useful tool in mushroom improvement by gene engineering.

Drug Resistance, Fungal↗
PubMed1999

[The immunodiagnosis and immunotherapy of tumors].

Recent studies into the immunology of tumors have allowed the oncological use of immunological methods to be substantially extended. This increases the potentialities of immunodiagnosis, primarily to identify a number of tumor-associated antigens, which has promoted early differential diagnosis and monitoring of the course of disease. On the other hand, diagnosis of immunodeficiencies in cancer patients has become better: a panel of monoclonal antibodies to differentiated immunocompetent cell antigens has been developed, their subpopulations revealed, the time course of whose quantitative composition correlates with the clinical course and prognosis of disease. Evidence for immunological disorders serves as a basis for using different immunotherapies in the complex treatment of cancer patients. Present-day oncological care uses a great variety of immunomodulators; however, preferable treatment includes cytokines (interferons, interleukins) whose application substantially enhances the efficiency of treatment in patients having certain tumors. Progress in biotechnology and gene engineering has contributed to the development of new trends in the therapy of tumors, namely the design of anticancer vaccines obtained by inserting the genes of cytokines and their receptors, tumor-associated antigens and other agents into the genome of a tumor cell. So far single clinical observations are yielding promising results.

Adult↗
PubMed2003

[Relativity betweeen expression level of potato virus X coat protein and the codon usage of wobble codon].

The relationship between the quantities of rare codons and the expression levels of foreign genes is very important in gene engineering. But up to now, there is few experiment on it in multi-cellular eukaryotes, especially in plants. To study the effect of rare codons on the expression level of potato virus X (PVX) coat protein (CP) in transgenic tobacco, some preferred codons in PVX coat protein gene were substituted with synonymous rare codons without changing the encoded amino acid sequence using site-specific mutation. The modified coat protein gene (cpm) and non-modified coat protein gene (cpw) were inserted into binary vector under the control of CaMV35S promoter, and these two plant expression constructs were transferred into tobacco (Nicotiana tabacum cv. Xanthi) genomes via Agrobacterium mediated method and transgenic plants were generated. Western blot and ELISA of these transgenic tobaccos showed that the expression level of modified CP is about one third to one sixth of that of non-modified CP. Northern blot analysis of RNAs from some transformants indicated that the transcriptional levels of cpm are the same as that of cpw, which implicated that the quantity of rare codons in foreign gene was a possible limited factor in foreign gene expression process. Changing the quantity of rare codons maybe an effective method for organism to control gene expression.

Amino Acid Sequence↗
PubMed2004

Wet wound healing: from laboratory to patients to gene therapy.

Wet treatment of wounds has been used as an "irrigation" method since the seventh century. We have developed the concept of an in vivo tissue culture that facilitates wound healing and allows tissue engineering. A transparent, flexible, round chamber provides the wet environment. This system heals clean wounds as fast or faster than any other method, with less scarring. It allows delivery of analgesics, antibiotics, growth factors, growth media, and cells into the chamber, becoming a platform for tissue engineering. Gene therapy of the wound can be done in the chamber with growth factor and other genes. A tetracycline switch allows precise timing and amounts of expression and provides the opportunity for sequential expression of genes delivered at the same time.

Epidermal Growth Factor↗
PubMed1994

Regulated immunoglobulin (Ig) RNA processing does not require specific cis-acting sequences: non-Ig RNA can be alternatively processed in B cells and plasma cells.

Alternative RNA processing of the heavy-chain immunoglobulin mu gene is regulated during B-cell maturation and requires competition between splice and cleavage-polyadenylation reactions that have balanced efficiencies. Studies with modified mu genes have failed to identify gene-specific sequences required for regulation. Thus, the only important feature for regulation may be the balanced competing splice and cleavage-polyadenylation reactions themselves. If this is so, then alternative RNA processing from any gene with similar competitive RNA processing pathways should also be regulated when expression is compared between B cells and plasma cells. To test this prediction, two nonimmunoglobulin genes engineered to have competing splice and cleavage-polyadenylation reactions were expressed in B cells and plasma cells. The ratios of alternative RNAs produced from both genes are different in the two cell types; like the mu gene, relatively more spliced RNA is produced in B cells than in plasma cells. Also, in a survey of mu gene expression in nine non-B-cell lines, only a T-cell line had an expression pattern similar to that of B cells; the expression patterns of all other lines resembled that of the plasma cells. Therefore, regulated mu RNA processing must be mediated by changes in general processing factors whose activity or abundance is regulated, most likely, in B cells.

Alternative Splicing↗
PubMed2003

[Construction and screening of TsF cDNA library specific for AchR].

OBJECTIVE: To construct the T suppress factor (TsF) cDNA library specific for AchR and screen out the positive clones of TsF. METHODS: The full-length cDNA was synthesized by the reverse transcripting from the TsF PolyA+mRNA, which had been extracted from the AchR specific suppressor lines (ARSL). The cDNA library was obtained using Lambda gt11 vector. Finally, this cDNA library was screened by Western blotting using Analysis of this cDNA library showed that titer of the monoclonal antibody specific for TCR-achain. RESULTS: recombinant bacteriophage was 1.4 x 10(7) pfu/ml, the rate of positive recombinant was 86.9%. The recombinant bacteriophage DNA were digested by EcoRI and electrophoresis analysis found that the main size of insertion was 0.8 to approximately 4.0 kb . As a result of screening, 27 positive clones were obtained. The positive recombinant bacteriophage DNA were digested by EcoRI, and electrophoresis analysis found that the size of insertion was 1 kb. Conclusion The results suggested that this cDNA library is available, the recombination phages can express TsF specific for AchR effectively and could be used to prepare the products of gene engineering of TsF.

Animals↗
PubMed1994

[Organ xenotransplantation using human complement regulatory factor gene].

For the success of xenotransplantation, hyperacute rejection must be controlled. Hyperacute rejection is induced by the activation of complement system via alternative pathway and/or classical pathway. Complement regulatory factors can inhibit activation of complement system via the both pathways. To overcome hyperacute rejection in organ xenotransplantation, gene engineering in the establishment of xeno-endothelial cells and transgenic animals (mouse and pig) using human complement regulatory factors are studied. In the near future, transgenic pig using human complement regulatory factor genes can be used as a potential donor in organ xenotransplantation.

Animals↗
PubMed1995

Tissue-specific expression of the gene for type I procollagen (COL1A1) in transgenic mice. Only 476 base pairs of the promoter are required if collagen genes are used as reporters.

Inconsistent data have been reported on the size of the promoter that is necessary for high levels of tissue-specific expression of the COL1A1 gene for type I procollagen. Some of the inconsistencies may be traced to the use of reporter gene constructs. Therefore, we prepared transgenic mice with modifications of the intact gene engineered so that the level of expression of the transgene could be assayed both as mRNA and protein that were similar to the products from the endogenous COL1A1 gene. The results with a mini-COL1A1 gene lacking 41 internal exons and introns indicated that the first intron and 90% of the 3'-untranslated region were not essential for tissue-specific expression. In a hybrid COL1A1/COL2A1 construct, a 1.9-kilobase 5'-fragment from the COL1A1 gene that contained only 476 of the promoter was linked to a promoterless 29.5-kilobase fragment of the human COL2A1 gene for type II procollagen. The hybrid COL1A1/COL2A1 construct was expressed as both mRNA and protein in tissues that normally synthesize type I procollagen but not type II procollagen. Apparently, 476 base pairs of the promoter are sufficient to drive tissue-specific expression of the COL1A1 gene and totally inappropriate expression of the COL2A1 gene.

Animals↗
PubMed1988

[Isolation and properties of genetic engineered human leukocyte interferons alphaI1 and alphaN].

Using controlled pore glass chromatography and immunoaffinity chromatography on monoclonal antibodies NK-2 immobilized on Sepharose 4B, the electrophoretically homogeneous interferons alpha N and alpha I1 were isolated from the biomass of gene-engineered Pseudomonas sp. strains. In terms of specific activity on human fibroblast diploid cells, interferon alpha I1 does not differ from interferon alpha A, whereas the specific antiviral activity of interferon alpha N is as low as 2.10(7) JU/mg. The procedures for immunometric assay of interferons alpha N and alpha I1 have been elaborated. Various monoclonal antibodies to interferon alpha A and to natural leucocyte interferon were analyzed; among those the antibodies specifically interacting with interferons alpha N and alpha I1 (but not with interferon alpha A) were identified.

Amino Acids↗
PubMed2001

Targeting of G(D2)-positive tumor cells by human T lymphocytes engineered to express chimeric T-cell receptor genes.

Genetic engineering of human T lymphocytes to express tumor antigen-specific chimeric immune receptors is an attractive means for providing large numbers of effector cells for adoptive immunotherapy while bypassing major mechanisms of tumor escape from immune recognition. We have applied this strategy to the targeting of a G(D2)-positive tumor, neuroblastoma, which is the commonest extracranial solid tumor of childhood. Chimeric immune receptors were generated by joining an extracellular antigen-binding domain derived from either of the 2 ganglioside G(D2)-specific antibodies sc7A4 and sc14.G2a to a cytoplasmic signaling domain. The variable domains of hybridoma antibody 14.G2a were cloned and selected using a phage display approach. Upon coincubation with G(D2)-expressing tumor cell targets, human T lymphocytes transduced with recombinant retroviruses encoding chimeric receptors based on sc14.G2a, but not sc7A4, secreted significant levels of cytokines in a pattern comparable to the cytokine response obtained by engagement of the CD3 receptor. T cells transduced with the sc14.G2a-based chimeric T-cell receptors also displayed specific lysis of G(D2)-positive neuroblastoma cells, which was blocked in the presence of monoclonal antibody 14.G2a. In the absence of nonspecific stimulation of transduced cells, their functionality declined over time and antigenic stimulation of the chimeric receptor alone did not induce commitment to proliferation. These results support the feasibility of redirecting human T lymphocytes to a tumor-associated ganglioside epitope but emphasize that successful chimeric receptor-mediated adoptive immunotherapy will require additional strategies that overcome functional inactivation of gene-modified primary T lymphocytes.

Cytokines↗
PubMed2005

Engineered riboswitches control gene expression by small molecules.

We have developed conditional gene expression systems based on engineered small-molecule-binding riboswitches. Tetracycline-dependent regulation can be imposed on an mRNA in yeast by inserting an aptamer in its 5'-untranslated region. Biochemical and genetic analyses determined that binding of the ligand tetracycline leads to a pseudoknot-like linkage within the aptamer structure, thereby inhibiting the initial steps of translation. A second translational control element was designed by combining a theophylline aptamer with a communication module for which a 1 nt slipping mechanism had been proposed. This structural element was inserted close to the bacterial ribosomal binding site at a position just interfering with translation in the non-ligand-bound form. Addition of the ligand then shifts the inhibitory element to a distance that permits efficient translation.

Base Sequence↗
PubMed2002

Cancer immunotherapy using gene-modified dendritic cells.

Gene-engineered dendritic cells (DC) are being tested in cancer immunotherapy. DC are the best equipped antigen-presenting cells (APC) to overcome tolerance/ignorance to self antigens presented by cancer cells. Genetic immunotherapy with DC engineered to express tumor antigens has the potential advantages of endogenous epitope presentation by both major histocompatibility complex (MHC) class I and II molecules. DC can also be gene-modified to express immunostimulatory molecules that further enhance their antigen-presenting function. Review of the literature provided 52 manuscripts where gene-modified DC were being tested in murine models of immunotherapy for cancer. Review of the antitumor effects of gene-modified DC in these preclinical studies provides valuable information on the optimal methods of gene transfer into DC, the schedule of administration, the route, dose and the underlying immunological mechanisms of the antitumor effects. These data may help in the translation of this promising approach to the clinic.

Adenoviridae↗
PubMed2004

[Potential of cdc25A-Fas chimeric expression vector in inducing apoptosis of Tca8113 cells in vitro].

BACKGROUND & OBJECTIVE: Previous studies have revealed the close relationship between Fas/FasL pathway and carcinogenesis of oral squamous cell carcinoma (OSCC). This study was designed to explore the potential of cdc25A-Fas chimeric expression vector in inducing apoptosis of human OSCC cell line Tca8113. METHODS: The 2 chimeric expression vector pAdTrack-CMV-cdc25A-Fas (pCCF), and pAdTrack-cdc25A-Fas (pCF) were constructed by gene engineering, pCCF, pCF, and the control plasmid pAdTrack-CMV were transfected into Tca8113 cells by liposome, respectively. The transfection efficiency was presented by the expression of the report gene, green fluorescence protein (GFP). The mRNA and protein levels of Fas were determined by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemistry methods. The apoptosis of transfected Tca8113 cells was analyzed by techniques of DNA agarose gel electrophoresis, TUNEL, Annexin V label, and flow cytometry (FCM). RESULTS: (1) The chimeric expression vectors pCCF, and pCF were successfully transfected into Tca8113 cells and the maximum transfection (15%) was observed at the 5th-7th day after transfection. (2) Up-regulation of Fas expression was detected at the 3rd day after transfection in pCCF, and pCF transfection groups. At 3rd, 5th, and 7th day after transfection, Fas protein was found to express on membrane and in plasma of transfected Tca8113 cells with distinct morphology of partial apoptosis. (3) From 2.5 days after transfection, early apoptosis had been observed in pCCF, and pCF transfection groups. At 3rd day, the increased apoptosis index (AI,=25%) of pCCF, and pCF transfection groups was significant higher than that of control group (P< 0.05), whereas there was no significant difference in AI between pCCF transfection group and pCF transfection group (P >0.05). (4) FCM analysis showed that the peak of GFP-expression cells was identical to that of the apoptotic cells. CONCLUSION: The cdc25A-Fas chimeric expression vector was able to initiate the apoptosis of Tca8113 cells by up-regulating Fas expression. This result indicated that the 27 bp cdc25A fragment can be designed as a cis-regulatory element to modulate the effects of C-Myc/Max in cellular proliferation and apoptosis.

Apoptosis↗
PubMed2004

[Antifreeze proteins and molecular genetic improvement in freezing resistance of plants].

Over the past several years, the proteins and genes associated with freezing resistance and molecular genetic improvement in freezing resistance of plants have been widely studied. The recent progress of research made at home and abroad on low-temperature-induced proteins and antifreeze proteins (AFPs) with thermal hysteresis (THA) and the identification and expression regulation of low-temperature-induced genes are reviewed in this paper. Recent advances in the approaches of gene engineering that have been successfully taken in the molecular improvement of plant freezing resistance are also reviewed. Emphasis is placed on the transformation and expression of the freezing resistance genes cloned from overwintering insects, polar fishes and plant materials. Finally, some unsolved problems and the trend of research on physiological function and mechanism of AFPs, and the application of modern biotechnology to molecular improvement in freezing resistance of plants are discussed.

Antifreeze Proteins↗